Significance Light microscopy advanced our understanding of cellular structure and associated functions. Research has shown that cellular events, such as signal transduction and gene transcription, require the assembly of proteins into specific macromolecular complexes. Traditional biophysical or biochemical methods have not provided direct access to interactions of protein partners in their natural environments but light microscopic techniques allow us to study molecules under physiological conditions. New imaging technologies, coupled with the development of new genetically encoded fluorescent labels and sensors, and the increasing capability of computer software for image acquisition and analysis, have enabled researchers to conduct more sophisticated studies of the functions and processes of protein molecules, ranging from gene expression to second-messenger cascades and intercellular signaling (DelPozo et al., 2002; Struck et al., 1981; Roessel and Brand, 2002; Ting et al., 2001). One highly sensitive and non-invasive method of protein molecular imaging is Forster (fluorescence) resonance energy transfer (FRET) microscopy. FRET is a distance-dependent physical process, where energy is transferred nonradiatively from an excited molecular fluorophore (donor) to another fluorophore (acceptor) by means of intermolecular longrange dipole-dipole coupling. FRET can accurately measure molecular proximity (1-10 nm), typically when donor and acceptor are positioned within the Forster radius (the distance at which half the excitation energy of the donor is transferred to the acceptor, ~3-6 nm). The efficiency of FRET is dependent on the inverse sixth power of intermolecular separation (Forster, 1965; Lakowicz, 1999; Stryer, 1978), making it a sensitive method for investigating a variety of biological phenomena that produce changes in molecular proximity (Cummings et al., 2002; Day et al 2003; Miyawaki et al., 1999; Wallrabe et al., 2003a). If FRET occurs, donor fluorescence is quenched and acceptor fluorescence is sensitized (increased) (Periasamy and Day, 2005). Co-localization of the donor- and acceptor-labeled probes can be seen within ~0.09 um and molecular associations at close distances can be verified.